Dubrovitsy, Podolsk District, Moscow Province, 142132 Russia, e-mail vijmikrob@mail.ru, DAP2189@gmail.com, asnd@mail.ru, elenagladyr@mail.ru, n_zinovieva@mail.ru Acknowledgements: Supported by Russian Science Foundation (project ¹ 15-16-00020) Received September 9, 2016 A b s t r a c t The development of microbial resistance to antibiotics determines the effectiveness of the veterinary measures. <...> The study and monitoring, well-designed treatment schemes are those measures that help to reduce the risk of spread of pathogenic and conditional-pathogenic microorganisms. <...> One of the problems in the dairy farming is mastitis of high producing cows which is mainly caused by Staphylococcus aureus. <...> Here we first report findings on the development of resistance in S. aureus strains isolated from milk of clinically healthy dairy cows during treatment with antibiotics of different classes. <...> The study was carried out using high yield holsteinized black-and-white cows (n = 1321, Kaluga region, 2016) which were vaccinated twice with anti-mastitis drug Mastivak («Ovejero», Spain). <...> Milk was sampled during the lactation period individually from each cow. <...> For the species identification of S. aureus isolates we used the following criteria: (i) morphology and microscopy of the colonies grown on the Baird Parker Agar medium («HiMedia Laboratories Pvt., Ltd», India); (ii) the presence of hemolysis zones on the Azide Blood Agar Pronadisa medium («Conda», Spain) supplemented with 5 % defibrinated sheep blood; (iii) a coagulation of dry citrated rabbit plasma (CJSC «EKOlab», Russia); (iv) biochemical characterization with API 20 STAPH panel («BioMerieux», France). <...> The sensitivity of the isolated microorganisms to ten antibiotics («Pharmacotherapy Research Center», Russia) including penicillin (PEN, 10, ED), oxacillin (OX, 10 μg), gentamicin (GN, 10 μg), erythromycin (ER, 15 μg), lincomycin (LN, 15 μg), rifampicin (RF, 5 μg), ciprofloxacin (CP, 5 μg), vancomycin (VA, 30 μg), fuzidin (FZ, 10 μg) and novobiocin (NB, 5 μg) was determined by disk diffusion method. <...> The majority of the strains were resistant to novobiocin (49.7 %) while the smallest part (2.6 %) exhibited resistant to vancomycin. <...> Importantly, the antibiotic resistance to next-generation antibacterial agents (novobiocin <...>